The rapid increment in rat hepatic mRNA-S14 after administration of T3 to hypothyroid rats makes this sequence an attractive model for studies of thyroid hormone action. A large body of physiological evidence indicates a strong association of S14 gene expression with lipogenesis. The purpose of the proposed extension is to achieve the goals of (1) characterizing the function and regulation of the S14 protein, and (2) defining the mechanism whereby T3 augments S14 gene transcription by analyzing the kinetics of the in vitro transcriptional assay. During the current grant period we have raised antisera to a synthetic peptide corresponding to a portion of the S14 protein, as deduced from the coding region of the mRNA-S14 sequence, which specifically recognize the protein on Western blots and in immunoprecipitation reactions. We propose lo utilize the sera during the proposed grant extension to purify the protein by affinity chromatography, to compare regulation of the protein and its mRNA employing a radioimmunoassay, and to probe for a role for the protein in lipogenesis by immune inactivation of it in both in an vitro lipogenesis system and in cultured hepatocytes. The rapidly of the response of S14 gene expression to T3 leads us to believe that these studies will provide insight into the mechanism whereby T3 acts to regulate lipogenesis. We also propose to focus on the mechanism underlying the rapid, antagonistic interaction of T3 and glucagon in the regulation of S14 gene transcription, assessed in a nuclear run-on assay. Our results indicate that small doses of T3 reverse the glucagon- mediated inhibition of S14 gene transcription within 5 min of T3 injection, and suggest the hypothesis that these stimuli may act to regulate the turnover of nascent primary transcripts prior to their entry into the nuclear precursor pool. We propose to critically assess the role of cAMP as a mediator of glucagon- induced inhibition of S14 gene transcription, and to look for a nuclear phosphoprotein exhibiting kinetics appropriate to those of the T3-glucagon interaction if such a role is documented. We will directly assess the potential roles of glucagon and T3 as modulators of degradation of nascent precursor mRNA-S14 transcripts in the nuclear run-on assay by pulse-chase. These studies are designed to examine the hypothesis that the nuclear T3-receptor complex acts as a negative control element by antagonizing stimuli which inhibit S14 gene expression.